ng into competents cells - (Sep/12/2008 )

Hi guys! I'm doing ligation reactions with different insert:vector ratios. which is the minimal or the highest amount in term of ng that I can use?I've not the sense of the measure..I use T4 ligase enzime in 10 ul of final volume for 16 h. Is also possible do the reaction at room temperature for all the week end?(somebody said me to do, but for me is very strange!).who has a good protocol, please give me some suggestion:thank u very much

-Fralab-

QUOTE (Fralab @ Sep 12 2008, 09:16 PM)

Hi guys! I'm doing ligation reactions with different insert:vector ratios. which is the minimal or the highest amount in term of ng that I can use?I've not the sense of the measure..I use T4 ligase enzime in 10 ul of final volume for 16 h. Is also possible do the reaction at room temperature for all the week end?(somebody said me to do, but for me is very strange!).who has a good protocol, please give me some suggestion:thank u very much

For vector:insert ratio, you can freely do it from 1:1 to 1:5. Usually i maintain it around 1:3. You must be remember that these ratio are the amount of dna but not the volume.

For the amount of plasmid, i maintained it between 50-400ng. Anyway i do calculation of plasmid to insert ratio and adjust their final volume to be 10ul (total reaction volume is 20ul)

For incubation time, i performed it in room temperature for about 1 hour. My labmate used to incubate overnight in 4oC. Both methods worked. Is there any special reason for incubating at room temperature for whole the weekend? Please share with me if you know. If there is not I assume it is an attitude of taking your experiment for granted which should not be practiced.